Exposure of PC12 cells to reagent peroxynitrite promotes the release of arachidonic acid (AA) mediated by activation of phospholipase A2 [Guidarelli, Palomba and Cantoni (2000) Br. J. Pharmacol. 129, 1539–1542]. We now present experimental evidence consistent with the notion that this response is not directly triggered by peroxynitrite but, rather, by reactive oxygen species generated at the level of complex III of the mitochondrial respiratory chain. In particular, superoxide (and not hydrogen peroxide) has a pivotal role in peroxynitrite-dependent activation of phospholipase A2. This observation was confirmed by results showing that superoxide, or peroxynitrite, promotes release of AA in isolated mitochondria. Consistently, the release of AA elicited by either peroxynitrite or A23187 in intact cells was shown to be calcium-dependent and differentially affected by phospholipase A2 inhibitors with different levels of specificity. In particular, the effects of peroxynitrite, unlike those of A23187, were both sensitive to low concentrations of two general phospholipase A2 inhibitors and insensitive to arachidonyltrifluoromethyl ketone, which shows some selectivity towards cytosolic phospholipase A2. In addition, peroxynitrite and A23187 synergistically enhanced the release of AA. Collectively, the above results demonstrate that peroxynitrite causes inhibition of complex III, followed by enforced formation of superoxides that stimulate the activity of a calcium-dependent PLA2 isoform, probably localized in the mitochondria.

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